The invention relates to a method for the metering and mixing of small quantities of liquid, to a device and to an apparatus for carrying out the method and to a use.
Diagnostic assays, in particular in the field of clinical chemistry and immunochemistry, are carried out in an automated manner to a large extent today. Defined volumes of sample liquid and reagents are pipetted into a cuvette or into the well of a microtiter plate and mixed in the corresponding automatic units. Subsequently, a first reference measurement is made in which, for example, the optical transmission through the cuvette is determined. After a certain reaction time between the sample and the reagents, a second measurement of the same parameter is made. The concentration of the sample with respect to a specific constituent or also only the presence of the constituent results by the comparison of the measured values. Typical volumes lie in sum at some hundred microliters, with necessary mixture ratios of sample to reagent being able to occur between 1:100 and 100:1. Optionally, a plurality of reagents can also be provided for mixing with a sample. In addition to the instruments just described for a high throughput, which are typically to be found in special laboratories, there are also endeavors to carry out assays in a decentral manner and without any large instrumental effort. It would be desirable in this connection if the “lab-on-a-chip” technology recently introduced could be used in which the processing of liquids on or in a chip be can carried out in an integrated manner. Assay times of less than one hour are desirable.
Microfluid systems are used, for example, for the movement of the liquids in which liquid is moved through electro-osmotic potentials, see for example Anne Y. Fu, et al. “A micro fabricated fluorescence-activated cell sorter”, Nature Biotechnology Vol. 17, November 1999, p. 1109 ff.
A method for liquid mixing in the microliter range is described in DE 103 25 307 B3 in which small liquid volumes are mixed in microtiter plates with the help of noise-induced flow. Another method for the generation of movement in small quantities of liquid on a solid surface is described in DE 101 42 789 C1. Here, a liquid is mixed or a plurality of liquids are mixed with one another with the help of surface sound waves.
In accordance with a method described in DE 100 55 318 A1, a quantity of liquid is placed onto a region of a substantially planar surface whose wetting properties differ from the surrounding surface such that the liquid preferably remains there, with it being held together by its surface tension. Movement of the quantity of liquid can be generated in this connection by the pulse transfer of a surface sound wave to the liquid.
In particular the integration of the metering and the mixing of the sample and the reagents in a cost-favorable lap-on-a-chip system is problematic. A homogeneous mixing of different quantities of liquid which are so small is difficult to realize.
It is necessary to define volumes of quantities of liquid precisely for the metering. This can be carried out geometrically, for example. For example, in an open system, the wetting properties of the surface can thus determine a volume, as is described in DE 100 55 318 A1. Here, the definition of the volumes takes place by hydrophilic and hydrophobic regions over the wetting angle on a substantially smooth surface. If a plurality of volumes were defined in this manner which should be brought to reaction, the volumes are moved toward one another to achieve this. On the movement on a surface, liquid residues or molecules of the analyte or of the reagent located in the liquid can remain stuck to the surface so that a volume loss or a reduction in concentration of unknown amount cannot be precluded by the movement. In addition, measures must be taken against evaporation which can in particular be problematic with longer assay times.
Other preparations use passages of defined cross-section which are filled with liquid in a capillary manner. If the liquid is an aqueous solution, a hydrophobic barrier which cannot be filled in a capillary manner is attached to the end of the passage. Furthermore, there is a lateral branch at this passage with a likewise hydrophobic surface which cannot be filled in a capillary manner. The cross-section and length of the passage between the hydrophobic barrier and the hydrophobic branch now determine a volume which can be separated and moved in a defined manner by pneumatic pressure through the branch (Burns et al., An integrated nanoliter DNA analysis device, Science 282, 484 (1998)). High costs arise by this type of volume definition due to the necessary wetting structuring of the surface (hydrophilic for the filling of the passage itself and hydrophobic for the barrier and the branch). In addition, it is necessary to work with air pressure, which requires corresponding devices. The passage cross-section must be small to permit the capillary filling of the measurement passage. Long passages are therefore necessary with large volumes in the range of some 100 microliters. This necessarily results in large unwanted interactions of the molecules in the liquid with the passage wall. An efficient mixing of a plurality of quantities of liquid is almost impossible in this geometry.
The term “liquid” in the present text includes inter alia pure liquids, mixtures, dispersions and suspensions as well as liquids in which solid particles are located, for example biological material. Liquids to be metered and to be mixed can also, for example, be two or more similar solutions which only differ by constituents dissolved therein which should be brought to reaction.